Antiviral immunity compromises the efficacy of adeno-associated virus (AAV) vectors used for gene therapy. This is well understood for the adaptive immune response. However, innate immune effectors like alpha-defensin antimicrobial peptides also block AAV infection, although their mechanisms of action are unknown. To address this gap in knowledge, we investigated AAV2 neutralization by human neutrophil peptide 1 (HNP1), a myeloid alpha-defensin, and human defensin 5 (HD5), an enteric alpha-defensin. We found that both defensins bind to AAV2 and inhibit infection at low micromolar concentrations. While HD5 prevents AAV2 from binding to cells, HNP1 does not. However, AAV2 exposed to HD5 after binding to cells is still neutralized, indicating an additional block to infection. Accordingly, both HD5 and HNP1 inhibit externalization of the VP1 unique domain, which contains a phospholipase A2 enzyme required for endosome escape and nuclear localization signals required for nuclear entry. Consequently, both defensins prevent AAV2 from reaching the nucleus. Disruption of intracellular trafficking of the viral genome to the nucleus is reminiscent of how alpha-defensins neutralize other non-enveloped viruses, suggesting a common mechanism of inhibition. These results will inform the development of vectors capable of overcoming these hurdles to improve the efficiency of gene therapy.

中文翻译：

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人 α-防御素中和 AAV2 的机制


抗病毒免疫会损害用于基因治疗的腺相关病毒 （AAV） 载体的疗效。这对于适应性免疫反应来说已经很好理解了。然而，像 α-防御素抗菌肽这样的先天免疫效应子也可以阻断 AAV 感染，尽管它们的作用机制尚不清楚。为了解决这一知识差距，我们研究了人中性粒细胞肽 1 （HNP1）（一种骨髓 α 防御素）和人类防御素 5 （HD5）（一种肠道 α 防御素）对 AAV2 的中和作用。我们发现两种防御素都与 AAV2 结合并在低微摩尔浓度下抑制感染。虽然 HD5 会阻止 AAV2 与细胞结合，但 HNP1 不会。然而，与细胞结合后暴露于 HD5 的 AAV2 仍被中和，表明感染受到了额外的阻断。因此，HD5 和 HNP1 都抑制 VP1 独特结构域的外化，该结构域包含内体逃逸所需的磷脂酶 A2 酶和进入核所需的核定位信号。因此，两种防御素都会阻止 AAV2 到达细胞核。病毒基因组向细胞核的细胞内运输中断让人想起 α-防御素如何中和其他非包膜病毒，这表明一种常见的抑制机制。这些结果将为能够克服这些障碍以提高基因治疗效率的载体的开发提供信息。